Pgf3alpha

ABSTRACT

THE PROSTAGLANDIN PGF3A, AND ITS SALTS ESTERS, AND ALKANATES ARE DISCLOSED. THESE NOVEL COMPOUNDS ARE USEFUL FOR A VARIETY IF PHARMACOLOGICAL PURPOSES, INCLUDING USE AS SMOOTH MUCLE STIMULANTS AND AS CARDIOVASCULAR AGENTS.

United States Patent Oflice PGF Sune Bergstrom and Jan Sjovall, both Kemiska Institutionen, Karolinska Institutet, Stockholm, Sweden No Drawing. Application Feb. 12, 1971, Ser. No.

115,111, now Patent No. 3,706,789, which is a con- 5 tinuation-in-part of application Ser. No. 203,752, June 20, 1962, now Patent No. 3,598,858, which is a continuation-impart of abandoned application Ser. No.

119,209, Apr. 9, 1962, which in turn is a continuationin-part of application Ser. No. 738,514, May 28, 1958,

now Patent No. 3,069,322. Divided and this application July 3, 1972, Ser. No. 268,439

Int. Cl. C07c 61/32, 69/74 US. Cl. 260-468 D 12 Claims ABSTRACT OF THE DISCLOSURE The prostaglandin PGF3,,, and its salts, esters, and alkanates are disclosed. These novel compounds are useful for a variety if pharmacological purposes, including use as smooth muscle stimulants and as cardiovascular agents.

CROSS REFERENCE TO RELATED APPLICATIONS DESCRIPTION OF THE INVENTION This invention relates to novel compositions of matter. One aspect of this invention is specifically concerned with novel organic compounds of the formula: 1

v wherein Z is -CH CH or cisCH=CH-- and R is hydrogen or a pharmacologically acceptable cation, said compound being essentially free of pyrogens, antigens, proteins, enzymes, cellular material, and other organic acids or salts thereof.

Another aspect of this invention is specifically concerned with novel organic compounds of the formula:

wherein Z is CH2-CH3- or cis--CH=CH--, wherein R is hydrocarbyl of not more than 1 carbon atoms, and wherein R is hydrogen or lower alkanoyl.

Patented Apr. 16, 1974 Another aspect of this invention is specifically concerned with novel organic compounds of the formula:

wherein Z is -CH CH or cisCH=CH-, wherein R is hydrogen or a pharmacologically acceptable cation, and wherein R is lower alkanoyl.

Included in Formula I are compounds of the formulas:

n on (IV) HO H \OH (V) Included in Formula II are esters and ester-trialkanoates of the acids of Formulas IV and V.

Included in Formula III are trialkanoates of the acids of Formulas IV and V. I

Molecules of the compounds encompassed by Formulas I, II, III, IV, and V each have several centers of asymmetry. Formulas I, II, III, IV, and V are intended to rep:

resent optically active compounds each with the same absolute configuration as optically active prostaglandin E (PGE), later named prostaglandin E (PGE and obtained from certain mammalian tissues, for example, sheep vesicular glands. See our said Pat. No. 3,069,332. See also later publications, for example, Bergstrom et al., J. Biol. Chem. 238, 3555 (1963), Bergstrom et al., 'Pharmacol. Rev. 20, 1 (1968), and references cited in those.

In Formulas I, II, III, IV, and V, a broken line attachment to the cyclopentane ring indicates a chain or group in alpha configuration, i.e., below the plane of the cyclopentane ring. A heavy solid line attachment to the cyclopentane ring indicates a chain in beta configuration, i.e., above the plane of the cyclopentane ring. The configuration of the side chain hydroxy in Formulas I, II, III, IV, and V is S. v

A systematic name for the compound of Formula IV is 7- [301,5 a-dihydroxy-2B-[ (3S) -3-hydroxy-transl-octenyl] cis-5-heptenoic acid. For convenience, this compound is designated PGF I A systematic name for the compound of Formula V is 7-[3u,5oc-dihydroxy-2fl-[(3S)-3-hydroxy-trans-l cis 5- octadienyl]-la-cyclopentyl]-cis-5-heptenoic acid. For convenience, this compound is designated PGF PGF and PGF were previously named bisdehydro- PGF and tetradehydro-PGF, respectively. See our said copending application Ser. No. 203,752.

With regard to Formula II, examples of hydrocarbyl of not more than 13 carbon atoms are al-kyl, e.g., methyl,

3 propyl, hexyl, decyl; cycloalkyl, e.g., cyclopropyl, 2-butylcyclopropyl, cyclobutyl, cyclobutylmethy, 3-pentylcyclobutyl, 2,2-dimethylcyclobutyl, cyclopentyl, 3-tert-buty1- cyclopentyl, 2-cyclopentylethyl, cyclohexyl, cyclohexymethyl; aralkyl, e.g., benzyl, phenethyl, l-phenylethyl, 2- phenylpropyl, 3-phenylbutyl, Z-(I-naphthylethyl), benzhydryl; aryl, e.g., phenyl, p-tolyl, p-ethylphenyl, p-tertbutylphenyl, l-naphthyl; and such unsaturated moieties as allyl, crotyl, and propargyl.

With regard to Formulas II and III, examples of lower alkanoyl are alkanoyl of 2 to 8 carbon atoms, inclusive, e.g., acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, and branched chain isomeric forms of those, e.g., isobutyryl and isovaleryl.

The novel compounds of Formulas I, II, III, IV, and V, i.e., PGF PGF and their salts, esters, trialkanoates, and ester-trialkanoates are extremely potent in causing stimulation of smooth muscle as shown, for example by tests on strips of guinea pig ileum, rabbit duodenum, or gerbil colon. These compounds are also highly active in potentiating other known smooth muscle stimulators, for example, oxytocic agents, e.g., oxytocin and the various ergot alkaloids including derivatives and analogs thereof, Accordingly, these novel Formulae I, II, III, IV, and V compounds are useful in place of or in combination with less than the usual amounts of these and other known smooth muscle stimulators whenever smooth muscle stimulation is needed to alleviate or prevent some physiological condition in mammals, including humans, useful domestic animals, pests, zoological specimens, and laboratory animals, for example, mice, rabbits, rats, and monkeys. For example, these compounds can be used to alleviate or prevent conditions of gastrointestinal atony in mammals, including humans, e.g., paralytic ileus following anesthesia and surgical operation or from other medical causes. For this purpose, the compound is administered parenterally, e.g., subcutaneously, intramuscularly, or by intravenous injection or infusion in a dose range 0.1 to 2 mg. per kg. of body weight per day, the exact dose depending on the age, weight, and condition of the patient or animals, and the frequency and route of administration. Small repeated doses are indicated when the aim is to prevent rather than alleviate the atony.

Another smooth muscle stimulatory area where these novel Formula I, II, III, IV, and V compounds are useful, especially that of Formula IV, is in the control or prevention of atonic uterine bleeding in mammals after abortion or delivery, to aid in the expulsion of the placenta, and during the puerperium. For this purpose, the compounds are administered by intravenous infusion immediately after abortion or delivery at a dose in the range about 0.1 to about 100 g. per kg. of body weight per minute until the desired effect is obtained. Subsequent doses are given by intravenous, subcutaneous, or intramuscular injection or infusion during puerperium in the range 0.1 to 2 mg. per kg. of body weight per day, again the exact dose depending on the age, weight, and condition of the patient or animal.

In still another smooth muscle stimulatory area, these novel compounds of Formulas I, II, IH, IV, and V, especially that of Formula IV, are surprisingly useful in place of oxytocin to induce labor in pregnant female animals, including man, cows, sheep, and pigs, at or near term, or in pregnant animals with intrauterine death of the fetus from about 20 weeks to term. For this purpose, the compound is infused intravenously at a dose of 0.1 to 100 g. per kg. of body weight per minute until at or near the termination of the second stage of labor, i.e., expulsion of the fetus. These compounds are especially useful when the female is one or more weeks post-mature and natural labor has not started, or 12 to 60 hours after the membranes have ruptured and natural labor has not yet started. An alternative route of administration is oral.

The novel compounds of Fo m IV, and

V, especially that of Formula IV, are useful for controlling the reproductive cycle in ovulating female mammals, including humans and animals such as monkeys, rats, rabbits, dogs, cattle, and the like. By the term ovulating female mammals is meant animals which are mature enough to ovulate but not so old that regular ovulation has ceased. For that purpose, PGF for example, is administered systematically at a dose level in the range 0.1 mg. to about 20 mg. per kg. of body weight of the female mammal, advantageously during a span of time starting approximately at the time of ovulation and ending approximately at the time of menses or just prior to menses. Intravaginal and intrauterine are alternative routes of administration. Additionally, expulsion of an embryo or a fetus is accomplished by similar administration of the compound during the first third of the normal mammalian gestation period.

The novel compounds of Formulas I, II, III, IV, and V are also useful in mammals, including man, as nasal decongestants. For this purpose, the compounds are used in a dose range of about 10 g. to about 10 mg. per ml. of a pharmacologically suitable liquid vehicle or as an aerosol spray, both for topical application.

The novel Formulas I, II, III, IV, and V compounds of this invention are used for the purposes described above in free acid form, in ester form, in trialkanoate form, in ester-trialkanoate form, or in pharmacologically acceptable salt form. When the ester form is used, the ester is any of those within the above definition of R However, it is preferred that the R moiety not contain olefinic or acetylenic unsaturation. More preferred are alkyl esters wherein the alkyl moiety contains one to 8 carbon atoms, inclusive. Especially preferred are alkyl of one to 4 carbon atoms, inclusive. Of those alkyl, methyl and ethyl are especially preferred for optimum absorption of the compound by the body or experimental animal system.

Examples of alkyl of one to 4 carbon atoms are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl. Examples of alkyl of one to 8 carbon atoms are those mentioned above, and also pentyl, hexyl, heptyl, octyl, and branched chain isomers thereof, e.g., 2-ethylhexyl.

With regard to the trialkanoate or ester-trialkanoate forms of Formulas I and II, the alkanoyl moiety is any of those within the definition of R However, acetyl is especially preferred for optimum absorption of the compound by the body or experimental animal system. In Formula II it is intended that all R be hydrogen or that all be alkanoyl.

Pharmacologically acceptable salts of these Formulas I, II, III, IV, and V compounds wherein R is hydrogen useful for the purposes described above are those with pharmacologically acceptable metal cations, ammonium, amine cations, or quaternary ammonium cations.

Especially preferred metal cations are those derived from the alkali metals, e.g., lithium, sodium and potassium, and form the alkaline earth metals, e.g., magnesium and calcium, although cationic forms of other metals, e.g., aluminum, zinc, and iron, are within the scope of this invention.

Pharmacologically acceptable amine cations are those derived from primary, secondary, or tertiary amines. EX- amples of suitable amines are methylamine, dimethylamine, trimethylamine, ethylamine, dibutylamine, triisopropylamine, N-methylhexylamine, decylamine, dodecylamine, allylamine, crotylamine, cyclopentylamine, dicyclohexylamine, benzylamine, dibenzylamine, a-phenylethylamine, p-phenylethylamine, ethylenediamine, diethylenetriamine, and like aliphatic, cycloaliphatic, and araliphatic amines containing up to and including about 18 carbon atoms as well as heterocyclic amines, e.g., piperidine, morpholine, pyrrolidine, piperazine, and loweralkyl derivatives thereof, e.g., l-methylpiperidine, 4-ethylmorpholine, l-isopropylpyrrolidine, Z-methylpyrrolidine,

1,4-dimethylpiperazine, 2-methylpiperidine, and the like, as well asamines containing"water-solubilizing'orhydro philic groups, e.g., mono-, di-, and triejthariolamin'e', ethyldiethanolamine, N-butylethanolamine, '2-amino lbutanol, Z-amino-Z-ethyl-1,3-propanediol, 2 amino 2,-methyl 1 propanol, tris(hydroxymethyl)aminomethane,' N-phenylethanolamine, N (p' tert-amylphenyl)diethanolamine, galactamine, N-methylglucamine, N m'ethylglucosamine, ephedrine, phenylephrine, epinephrine, procaine, and the like. i i Examples of suitable pharmacologically acceptable quaternary ammonium" cations are 'f tetr'amethylammonium, tetraethylai'rimonium, 'benzyltrimethylammoiiium, phenyltriethlammonium, and the like.

When the novel Formula LII, III, IV, and V com,- pounds areused for intravenous injection or infusion, sterileaqueous isotonic solutions are preferred. For that purpose, it is preferred becauseof increas cdjwater solubility that a Formula IV or V acid or salt be'use'd, For subcutaneous or intramuscular injection, sterile'solutions or suspensions of the acid, salt, ,ester', or'alkanoate in aqueous or non-aqueous media are used. Tablets, capsules, and liquid preparations such. as syrups,elixers,,and

simple solutions, withthe usual pharmaceutical carriers are used for oral or sublingual administration. For rectal, vaginal, .or intrauterine administration, suppositories, lavage and douche preparations, and solutions as such or contained in a sponge, all prepared by methods known in the art, are.used. l I I PGF is prepared from the carboxylic acid known as PGE PGF is prepared from the carboxylic acid known as PGE PGE and PGE were previously designated bisdehydro-PGE and tetradehydro-PGE respectively, and the preparation of both of these reactant acids is described in our said copending application Ser. No. 203,- 752. The structural formulas of'PGE (VI) and PGE3 (VII) 'arejas follows: I W

v 11' 'OH" vnj I In these transformations "of P-GE and' -PGEQ-"tO PGFQ,

and PGF the ring carbony}, of the PG-E-type reactant 'is reduced to a secondary alcohol groupwith sodium boro hydride in the presenceof "an inert "diluenty advanta geously, methanol. A mixture of-two'f'isomeric secoridaly alcohols is produced by eachofthesering' carbonyl 're ductions.. For example,ring carbonyl reduction of PGE .gives a mixture of PGF ,"(Formula IV)" and'theisomeric compound known as PGFwfThelatter compound "has the structural formula; I

H .t In a similar manner, ring carbonyl reduction gives a mixture of PG-F and 2GB For these ring. carbonyl reductions; .a-.cold' solution .9: suspension of sodium borohydride in methanol" is added of PGE:

. 6 to. a cold solution of the PGE-type reactant in methanol. The mixture is maintained cold for about 10 to 60 minutes, and 'is then'rnai'ntained at about C. for about one to two hours. The resulting mixture of PGF -type andfPGF, type products is then isolated and separated into its components by chromatography as described here inafter. About three parts by weight of sodium borohydride isv used for each part by weight of the PGEv-type reactant. Suitable amounts of methanol are about 100ml. for each gram of the PGE-type reactant and about 100 ml. for each gram of the sodium borohydride. A suitable cold temperature is about 0 C. The acid of Formula IV, i.e., PG-F is also obtained from'the lung tissues of sheep and pigs. Procedures for doingthat are set forth in the examples, below.

Subsequent to our invention of the novel acid of Formula V, i.e., PGE that compound was obtained from bovine lung tissues. See Biochim., Biophys. Acta, 84, 707 (1964).

Q As mentioned above PGF and PGF are prepared by sodium borohydride reduction of PGE and PGE res'pectively, and PGF and PGF35 are also produced in those reactions. The PG-E-type reactants and the PGF,-

7 type byproducts have difierent spectra of biological activity than the desired PGF,,-type products. For example, PG'E and PGF are highly active in lowering arterial blood pressure, while PGF is not. When PGF and PGF are'prepared by these borohydride reductions and are to be used for one or more of the above-described pharmacological purposes, it is desirable that they be made essentially free of these possible contaminating acids or salts thereof. By the term essentially free here is meant a' degree of freedom from these other prostag'landihd-li k'e acids and salts such that the PGF and the PGF and their salts are suitable for the intended pharmacological uses, including parenteral administration to humans.

' Also as mentioned above, P'GF and PGF are obtainable from various mammalian lung tissues. These lung tissues contain many other components, i.e., pyrogens, antigens, proteins, enzymes, cellular material, and various other org anic acids and salts thereof. When PGF and PGFg,," arerobt'ained from these lung tissues and are to be used for one or more of the above-described pharmacologicalpurposes, it isnecessary that they be made essentially freeof these other lung' tissues components, i.e., pyrogens, antigens, proteins, enzymes, cellular material, and: other organic acids or salts thereof. By the term essentially free here is meant a degree of freedom from these impurities such that the PGF and the PGF are suitable for their intended pharmacological uses, includ ing parenteral administration to humans. This definition of degree of purity will include PGF and PGF prepared either by borohydride reduction of PGE and PGE respectively, or from mammalian lung tissues, since the PGE and PGE reactants and the PGF and PGF by; products 'in the borohydride reduction are included in the term other organic acids and salts thereof.

When PGF and PGF are purified to the above-defined l degr ee, of purity, they are considered to be essent'ially. pure and'useable for all of the above-described pharmacological purposes.

q When PGF and PGF are purified to the above-defined degree of purity, they will exhibit substantially ideal 6'5 curves on partition chromatography using an ethylene chloridezheptanezacetic acidzwater '(15:15:6:4) solvent system. By the term substantially ideal is meant curves typical of the essentially pure compounds.

When hydrocarbyl ester of PGF or PGF (Formula 11, R hydrocarbyl and all R '=H) is desired for one ormore oi the above-described pharmacological purposes, the RGF or PGF is esterified by procedures known in the art. vIllustratively, the alkyl esters are prepared by' reaction of the acid with the appropriate diazohydrocar: bon. For example, when diazomethane is used, the methyl esters are produced. Similar use of diazoethane, diazobutane, and 1-diazo-2-ethylhexane, for example, gives the ethyl, butyl, and 2-ethylhexyl esters, respectively.

Esterification with diazohydrocarbons is carried out by mixing a solution of the diazohydrocarbon in a suitable inert solvent, preferably diethyl ether, with the acid reactant, advantageously in the same or a different inert diluent. After the esterification reaction is complete, the solvent is removed by evaporation, and the ester purified if desired by conventional methods, preferably by chromatography. It is preferred that contact of the acid reactants with the diazohydrocarbon be no longer than necessary to effect the desired esterification, preferably about one to about ten minutes, to avoid undesired molecular changes. Diazohydrocarbons are known in the art or can be prepared by methods known inthe art. See, for example, Organic Reactions, John Wiley .& Sons, Inc., New York, N.Y., vol. 8, pp. 389-394 (1954).

An alternative method for esterification of the carboxyl moiety of these PGF -type acids comprises transformation of the free acid to the corresponding silver salt, followed by interaction of that salt with an alkyl iodide. Examples of suitable iodides are methyl iodide, ethyl iodide, butyl iodide, isobutyl iodide, tert-but'yl iodide, and the like. The silver salts are prepared by conventional methods, for example, by dissolving the acid in cold dilute aqueous ammonia, evaporating the excess ammonia at reduced pressure, and then adding the stoichiometric amount of silver nitrate.

When a trialkanoate of a Formula II ester or aFormula III acid is desired for one of the above-described pharmacological purposes, that is prepared by reacting the trihydroxy ester or trihydroxy acid with an alkanoic anhydride corresponding to an alkanoic acid of 2 to 8 carbon atoms, inclusive. Examples of these anhydrides are acetic anhydride, propionic anhydride, buty'ric anhydride, valeric anhydride, hexanoic anhydride, hepta'noic anhydride, octanoic anhydride, and isomeric formsof those. 1

This reaction leading to these trialkanoates'is advan-' tageously carried out by mixing the hydroxy compound and the acid anhydride, preferably in the presenceof 'a' tertiary amine such as pyridine or triethylamine. A.substantial excess of the anhydride is used, preferably about 10 to 10,000 moles of anhydride per mole of the hydroxy compound reactant. 'Ihe'excessanhydride serves as a reaction diluent and solvent. An inert organic'diluent, for example, dioxane, can also be added. Itis preferredto use enough of the tertiary amine to'neutraliize thecar boxylic acid produced by the reaction, as well as any free carboxyl groups present in the hydroxy compound reactant. p v, f

The reaction is preferably icarried outin'the'rang'e about to about 100 C. The necessary reaction'time will depend on such factors as the reaction temperature, and the nature of the anhydride and tertiaryamineireactants. With acetic anhydride, pyridine, and a"25,'C'. reaction temperature, a 12 to 24-hour reaction time is used. i, The'desired trialkanoate is isolated from the reaction mixture by conventional methods. For example the'excess anhydride is decomposed'wi'th water, and the resulting mixture acidified and then extracted with a solvent such as diethyl ether. The desiredtrialkanoate is recovered from the diethyl ether extract by evaporation. The tri alkanoate is then purified by conventional methods, ad-' vantageously by chromatography. j r r The 'Formula I, III, IV, and V PGFa-type acids and alkanoates are transformed to pharmacologically accept able salts by neutralization with appropriate'amounts of the corresponding inorganic or organic'base,";exarnples of which correspond to the cations andamines listed above. These transformations are carried outbyiava'riety of procedures known in the art to be generally useful for the preparation of inorganic, i.e., metal or ammonium,

' volatile amines.

salts, amine acid addition salts, and quaternary ammonium salts,' The choice of procedure depends in part upon the solubility characteristics of the particular salt to be prepared. In the case of the inorganic salts, it is usually suitable to dissolve the acid in water containing the stoichiOmetric'amount of a hydroxide, carbonate, or bicarbonate corresponding to the inorganic salt desired. For example, such use of sodium hydroxide, sodium carbonate, or sodium bicarbonate gives a solution of the sodium salt'of the prostaglandin derivative. Evaporation of the water or addition of a water-miscible solvent of moderate polarity, for example, a lower alkanol or a lower alkanone, gives the solid inorganic salt if that form is desired.

To produce an amine salt, the acid is dissolved in a suitable solvent of either moderate or low polarity. Examples 'of the former are ethanol, acetone, and ethyl acetate. Examples of the latter are diethyl ether and benzene. At least a stoichirnetric amount of the amine corresponding to the desired cation is then added to that solution. If the resulting salt does not precipitate, it is usually obtained in solid form by addition of a miscible diluent of low polarity or by evaporation. If the amine is relatively volatile, any excess caneasily be removed by evaporation. It is preferred to use stoichiometric amounts of the less Salts wherein the cation is quaternary ammonium are produced by mixing the acid with the stoichiometric amount of the corresponding quaternary ammonium hydroxide inwater solution, followed by evaporation of the water.

v The invention can be more fully understood by the following examples.

A-solution of 100 mg. of PGE dissolved in 10 m1. of methanol is cooled in an ice bath. A chilled solution of 300 mg. of sodium borohydride in 35 ml. of methanol is added. After 20 minutes at 0 C., the mixture is left at room temperature for one hour. Water is added and most of the methanol is taken'oifin vacuo'. After acidification with hydrochloric acid, the aqueous phase is extracted three times with ether,and/the combined ether extract is washed with water and brought to dryness at room temperature. The residue is subjected to reversed phase partition chromatography on 18 g. hydrophobic kieselguhr (treated with chlorornethylsilane), using 43 percent aqueous methanol as the mobile phase and equal parts of isooctanol and chloroform as the stationary phase. The dried ether extract is placed on the column with 16 ml. of the stationary phase and developed with 1200 ml. of mobile phase. The progress of the chromatography is followed by frequent microtitration of eluate fractions, using 0.02 N- aqueous sodium hydroxide solution. Two peaks of acidity ;in the series of eluate fractions are observed.

" I-heeluate fractions included in the major area of each eluateseto flowfrom the column is PGF The paper chromatographic-mobilities relative to PGE, on descending paper chromatography with ethylene chloride-heptane (1:1) as moving phase and aqueous acetic acid as stationary phase are PGE PGE (0.90),

PGF (0.60), and en 0.39 The PGF obtained by this process is. sufficiently pure to give a substantially ideal curve on partition chromatography, using an ethylene chloride:heptane;acetic-acidzwater (15:15 26:4) solvent system, that is, "a cui ve typical of the essentially pure compound.

Exam IeZPG sa Jjollowing the procedure 'of Example 1 but replacing the PG E' with PGEg, a mixture of PGF;,, and PGF is Example PGF Freeze-dried sheep lungs (12 kg.) are suspended in distilled water, using four liters per-kilogram of dried glands. After fifteen minutes, twelve liters. of 95 percent ethanol are added. The minced glands are stirred mechanically for about one hour, and thenleft to sediment over night. The supernatant, clear "ethanol solution, is decanted, and the insoluble residue is strained through cheesecloth and filtered. The supernatant and filtrate are a combined and evaporated in vacuo to about 5 the original volume, i.e., to about three liters. This crude extract is itself extracted with about three liters of ether. The water phase is then acidified to pH 3.5 and extracted again with three liters of etherand then twicewith 1.5 liters of ether. The combined ether extracts are extracted six times with volume or about 2.25 liters of0.2-molar phosphate buffer of pH 8., During the first extraction, the pH of the buffer has to be adjusted back to pH 8 with two normal sodium carbonate. The combined buffer phases are acidified to pH 3 with 6 normal hydrochloric acid and extracted with 1 volume, i.e., about 13.5 litersof ethyl acetate, then extracted three additional times each with seven liters of ethyl acetate. The ethyl acetate extracts are combined and washed until free of chloride ions with small portions of water, each Water portion being passed through a second ethyl acetate phase. The ethyl acetate is evaporated in vacuo, leaving a solid residue. The dried residue is then taken up in petroleum ether and extracted three times with an equal volume of 57 percent aqueous ethanol. The aqueous ethanol extract is concentrated in vacuo to remove the ethanol, and the aqueous phase is extracted with ether and the ether extract is evaporated to dryness. The crude extract thus obtained is then partitioned on a partition column using 50 percent aqueous methanol as the mobile phase and a 50-50 mixture of isooctanol and chloroform as the stationary phase. The support is finely-divided, low-temperature polyethylene. Seven grams of the crude extract is put on the column with 67 ml. of stationary phase on 100 g. of the support. The column is then developed with 2200 m1. of mobile phase. The peak of physiological activity appears at about 1500 ml. The 700 to 2200 ml. fractions are pooled. The pooled fractions are concentrated to an aqueous phase, extracted with ether, and the ether extract evaporated to dryness. Two hundred and fifty mg. of this extract is put on a column of 45 g. of hydrophobic diatomite (kieselguhr treated with chloromethylsilane) saturated with 40 ml. of stationary phase (isooctanol-chloroform, 1: 1) and developed with 2200 ml. of mobile phase (47.5 percent aqueous methanol). The peak of activity as determined by the microtitration and physiological activity appears at about 850 ml.

PGF is isolated from the 850 ml. peak fractions by concentration to aqueous under reduced pressure, extraction with ether, and evaporation of the extract. The PGF so obtained is suificiently pure to give a substantially ideal curve on partition chromatography using the same ethylene chloride:heptane:acetic acidzwater (15:15: 6:4) solvent system used to show the purity of the PGF,, obtained according to Example 1. Thus, the PGF- obtained by the above extraction process is essentially pure and is essentially free from pyrogens, antigens, proteins, enzymes, cellular material, and the other organic acids or salts thereof present in the sheep lung tissues. This PGF also exhibits the same mobility relative to PGE and P6 17 during paper chromatography as the PGFQ, prepaled a cdi'diilg to Example 1. i V

Example 4 'PGF Following the procedure of Example 3 but replacing the sheep lung with pig lungs, PGF- of the same degree of purity is obtained.

Example 5 PGF methyl ester To a dry ether solution of one milligram of PGF is added a slight excess of diazomethane prepared in ether from four micromoles of nitrosomethylurethane. The reaction mixture is allowed to stand about 5 minutes, and the ether and excess diazomethane are then distilled oif. On'dist'illation to dryness, PGF methyl ester is obtained.

Example 6 PGF methyl ester,

Following the procedure of Example 5, PGF is transformed to its methyl ester.

Example 7 methyl ester triacetate Example 8 PGF methyl ester triacetate Following the procedure of Example 7, PGF methyl ester is transformed to the triacetate.

The methyl esters and methyl ester triacetates of Examples 5, 6, 7, and 8 have the following retention times:

Methyl Methyl ester P GFa-type ester triacetate PGFzm 0.91 0.99 PGFaa 0. 91 0. 96

Conditions: Flash heater at 200 0.; Column temperature 200 C.; Column pressure 1.0 kg./cm. Column 6 ft. x 5 mm. packed with 0.5% QF-l (Dow Corning Corp.) on Gas Chrom P (Applied Science Laboratory, Inc.), as described by Vanden Heuvel et al., J. Am. Chem. Soc. 83,1513 (1961).

Following the procedures of Example 5 but replacing diazomethane with diazoethane, diazobutane, l-diazo-Z- ethylhexane, cyclohexyldiazomethane, and phenyl-diazomethane, and using PGF and PGF each in turn, there are obtained the ethyl, butyl, 2-ethylhexyl, cyclohexylmethyl, and benzyl esters of each of PGF and PGF Also following the procedure of Example 7 but using propionic anhydride, isobutyric anhydride, and hexanoic anhydride each in place of acetic anhydride, and using each in turn PCP- PGF and the methyl, ethyl, butyl, Z-ethylhexyl, cyclohexylmethyl, and benzyl esters of each of PGF and PGF there are obtained the tripropionates, triisobutyrates, and trihexanoates of each of PGF, acids and esters. Also following the procedure of Example 9, the triacetates of these other PGF, esters are obtained.

What is claimed is:

1. A compound of the formula:

11 wherein R is hydrocarbyl of not more than 13 carbon atoms, and wherein all R are hydrogen orall R are lower alkanoyl.

2. A compound according to claim 1 wherein R is alkylof one to 8 carbon atoms, inclusive, and all R are hydrogen.

3. A compound according to claim 1 wherein R is methyl and all R are hydrogen. I

4. A compound according to claim l wherein R is alkyl of one to 8 carbon atoms, inclusive, and wherein all R are lower alkanoyl.

5. A compound according to claim 1 wherein R is alkyl of one to 8 carbon atoms, inclusive, and wherein all R are acetyl.

6. A compound according to claim 1 wherein R is 15 methyl and all R are acetyl.

7. A compound of the formula:

R4 is wherein R is hydrogen or a pharmacologically acceptable cation, said compound being essentially free of pyrogens, antigens, proteins, enzymes, cellular material, and other organic acids or salts thereof.

11. A compound according to claim 10 wherein R is hydrogen, said compound being 7-[3a,5a-dihydroxy-2fl- [(3S)'-3-hydroxy-trans-1,cis-5-octadienyl] 1a cyclopentyl]-cis-5-heptenoic acid.

12.- A compound according to claim 10 wherein R is a pharmacologically acceptable cation, said compound being a salt of 7-[3a,5a-dihydroxy-2B-[(3S)-3-hydroxytrans 'lLcis-S-Octadienyl]-1u-cyclopentyl]cis-5-heptenoic acid.

' References Cited Linn et a1. Biochem. Pharmacology 8, 339 (1969).

LORRAINE A. WEINBERGER, Primary Examiner R. GERSTL, Assistant Examiner US. Cl. X.R.

260211 R, 247.2 R, 268 R, 293.65, 326.3, 410, 429.9, 430, 439, 448 R, 488 R, 501.1, 501.15, 510.17, 501.2, 514D UNITED STATES PATENT OFFICE Page 1 of 2 CERTIFICATE OF CORRECTION Patent No. 3: 79 Dated p l 97" Q Inventor(s) Sune Bergstrom and Jan Sjoval l It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, line 18, "alkanates should read alkanoates .line 19, "variety if pharmacological" should read variety of pharmacological l ine 71, "more than 1 carbon atoms," should read more than 15 carbon atoms, Column 2, lines 4-15,

Formula l I, that portion of the formula reading R4O\ R4=O\\ I I Q should read Ck R 0 R4 find H H =C should read =C (CH -C0OR (CH -CO0R l V, and V V, and V compounds Qlolumn 4, lines 51-52, "Formulas I, l

l compounds" should read Formula I, l

l l l Patent No.

. Inventor s) Dread i ng [SI-ALI UNITED STATES PATENT OFFICE Page 2 of 2 Dated April 97 Sune Bergstrom and Jan Sjoval l W should read I/ L H 0H ,4 new:

RUTH MASON A nesting Officer It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 12, Formula, Claim 10, that portion of the formula Signed and Scaled this Eighteenth Day of April 1978 LITRELLE F. PARKER Auing Commissioner of Patents and Trademarks 

